Methods for determining total body skeletal muscle mass

ABSTRACT

The present invention is based on the finding that enrichment of D3-creatinine in a urine sample following; oral administration of a single defined dose of D3-creatine can be used to calculate total-body creatine pool size and total body skeletal muscle mass in a subject. The invention further encompasses methods for detecting creatinine and D3-creatinine in a single sample. The methods of the invention find use, inter alia, in diagnosing disorders related to skeletal muscle mass, and in screening potential therapeutic agents to determine their effects on muscle mass.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/098,217, filed Apr. 13. 2016, which is a continuation of U.S. patentapplication Ser. No. 14/363,779, Internationally filed Dec. 6, 2012, nowabandoned, which is a U.S. National Phase Patent Application under 35U.S.C. §371 based on international Application No. PCT/US2012/068068,filed Dec. 6, 2012, which claims the benefit under 35 U.S.C. §119(e) ofU.S. Provisional Application No. 61/567,952, filed Dec. 7, 2011, andU.S. Provisional Application No. 61/708,013, filed Sep. 30, 2012, thedisclosures of which are hereby incorporated by reference in theirentirety.

FIELD OF THE INVENTION

This invention relates to methods for determining the total body poolsize of creatine and total body skeletal muscle mass in a subject by theuse of an orally administered tracer dose of D3-creatine, andencompasses improved methods for determining the concentration ofcreatinine in a biological sample.

BACKGROUND OF THE INVENTION

Skeletal muscle plays a central role in metabolic adaptations toincreasing and decreasing physical activity, in disease (e.g. cachexia),in obesity, and in aging (e.g. sarcopenia). Sarcopenia is described asthe age-associated loss of skeletal muscle (Evans (1995) J. Gerontal.50A:5-8) and has been associated with mobility disability (Janssen andRoss, (2005) J. Nutr. Health Aging 9:408-19) and greatly increasedhealthcare costs for elderly people (Janssen et al. (2004) J. Am.Geriatr. Soc. 52:80-5). Loss of skeletal muscle with advancing age isassociated with decreased energy requirements and concomitant increasein body fatness, weakness and disability, insulin resistance and risk ofdiabetes. Loss of skeletal muscle associated with an underlying illness(cachexia) is associated with a greatly increased mortality (Evans(2008) Clin. Nutr. 27:793-9).

Because of the important role total body skeletal muscle mass plays inaging and disease, there is an effort in the pharmaceutical arts toidentify therapeutic agents that will stimulate muscle protein synthesisand increase muscle mass. However, current methodologies forquantification of muscle synthesis and muscle mass often involveinvasive procedures (e.g. muscle biopsies) or rely on expensiveequipment (i.e. DEXA, MRI, or CT) that provides only indirect data onwhole body muscle mass. Because of these limitations, no method isroutinely used in the clinic for estimation of skeletal muscle mass, andno diagnostic criteria for estimates of muscle mass have been produced.As a result, there is a no straightforward way to determine the effectsof potential therapeutic agents on muscle protein synthesis mass.

Accordingly, there remains a need in the art for reliable,easily-performed, noninvasive measurements of total body skeletal musclemass.

BRIEF SUMMARY OF INVENTION

The present invention is based on the finding that steady-stateenrichment of D3-creatinine in a urine sample following oraladministration of a single defined tracer dose of D3-creatine can beused to calculate total-body creatine pool size and skeletal muscle massin a subject.

The invention is further based on the finding that the concentration ofcreatinine in a biological sample can he determined by measuring theconcentration of creatinine M+2 isotope and dividing this concentrationby a dilution factor, where the dilution factor is the ratio of theconcentration of creatinine M+2 to the concentration of creatinine M+0in the biological sample. Determining the creatinine concentration in abiological sample according to these improved methods allows for thesimultaneous measurement of the concentration of creatinine andD3-creatinine in a single sample using widely-available instrumentation.Accordingly, this improved detection method will facilitate thewide-spread adaptation of the present methods for use in determiningskeletal muscle mass in patients.

Accordingly, in one aspect the invention provides a method fordetermining the total body skeletal muscle mass in a subject, where themethod comprises the steps of:

(a) orally administering 10-200 mg D3-creatine or a salt or hydratethereof to the subject;

(b) allowing at least 12 hours to elapse after the administration of theD3-creatine;

(c) obtaining a biological sample from the subject,

(d) determining the concentration of creatinine and D3-creatinine insaid biological sample;

(e) using the creatinine and D3-creatinine concentrations determined instep

(f) to calculate the total body skeletal muscle mass of the subject.

In particular embodiments, the biological sample is a urine sample.

In certain embodiments, the concentration of creatinine andD3-creatinine in the urine sample is determined by HPLC/MS/MS.

In another aspect, the invention provides a method of determining theconcentration of creatinine in a biological sample from a subject, saidmethod comprising the steps of

(a) obtaining a biological sample from the subject;

(b) analyzing the biological sample to determine the peak area of thecreatinine M+2 isotope peak for the biological sample;

(c) comparing the peak area determined in step (b) to a calibrationcurve generated using D3-creatinine to determine the concentration ofthe creatinine M+2 isotope in the biological sample;

(d) dividing the concentration obtained in step (c) by a dilutionfactor, where the dilution factor is the ratio of the concentration ofcreatinine M+2 to the concentration of creatinine M+0 in the biologicalsample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Urinary D3-creatinine enrichment and total body creatine poolsize in growing rats. (A) Urinary D3-creatinine enrichment (determinedby isotope ratio mass spectrometry) in 9 week-old (mean body weight304±11 g, n=10) and 17 week-old (mean body weight 553±39 g, n=10) ratsat the indicated time after a single oral 0.475mg dose of D3-creatine,showing achievement of isotopic steady state by 48 h, and clearseparation of growing rat age groups (P<0.001 between groups at alltimes; within groups, the difference between 48 and 72 h is notsignificant; 2-factor ANOVA and Student's t test). (B) Creatine poolsize calculated from 72 h urinary D3-creatinine enrichments for the ratgroups in FIG. 1, showing clear separation of age groups (p<0.0001).

FIG. 2. Correlation between Lean Body Mass by Quantitative MagneticResonance and total body creatine pool size, adjusted for age effect,for the rat groups in FIG. 1 (r_(all rats)=0.69; P<0.001).

FIG. 3. Even within the rat groups of different age from FIG. 1, thereis a significant correlation of creatine pool size and lean body mass byeither quantitative magnetic resonance (left) or DEXA (right).

FIG. 4. Significant correlation between lean body mass determined byquantitative magnetic resonance and creatine pool size determined byD3-creatine dilution in 22 week-old rats (n=10 per group) treated theprevious two weeks with either vehicle or dexamethasone (P<0.001 andP=0.01, respectively).

FIG. 5. Correlation between lean body mass determined by quantitativemagnetic resonance and total-body creatine pool size determined byD3-creatine dilution for all 40 rats used in the two cross-sectionalstudies (y=0.20x+91.6; r=0.9517; P<0.0001).

FIG. 6. This figure shows a flow chart for one embodiment of the methodof determining total body skeletal mass.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the finding that enrichment ofD3-creatinine in a urine sample following oral administration of asingle defined dose of D3-creatine can be used to calculate total-bodycreatine pool size and skeletal muscle mass in a subject. Accordingly,the invention provides a non-invasive, accurate method of determiningtotal body skeletal muscle. The methods of the invention find use, interalia, in diagnosing and monitoring medical conditions associated withchanges in total body skeletal muscle mass, and in screening potentialtherapeutic agents to determine their effects on muscle mass.

According to the method, D3-creatine is orally administered to asubject. Although the present is not limited by mechanism, it isbelieved that the D3-creatine is rapidly absorbed, distributed, andactively transported into skeletal muscle, where it is diluted in theskeletal muscle pool of creatine. Skeletal muscle contains the vastmajority (>than 98%) of total-body creatine. In muscle tissue, creatineis converted to creatinine by an irreversible, non-enzymatic reaction ata stable rate of about 1.7% per day. This creatinine is a stablemetabolite that rapidly diffuses from muscle, is not a substrate for thecreatine transporter and cannot be transported back into muscle, and isexcreted in urine. As a result, once an isotopic steady-state isreached, the enrichment of a D3-creatinine in spot urine sample after adefined oral tracer dose of a D3 creatine reflects muscle creatineenrichment and can be used to directly determine creatine pool size.Skeletal muscle mass can then be calculated based on known musclecreatine content.

Accordingly, in one aspect the invention provides a method ofdetermining the total body skeletal muscle mass in a subject, where themethod comprises the steps of:

-   -   (a) orally administering 10-200 mg D3-creatine or a salt or        hydrate thereof to the subject;    -   (b) allowing at least 12 hours to elapse after the        administration of the D3-creatine;    -   (c) obtaining a urine sample from the subject,    -   (d) determining the concentration of creatinine arid        D3-creatinine in said urine sample;    -   (e) using the creatinine and D3-creatinine concentrations        determined in step    -   (f) to calculate the total body skeletal muscle mass of the        subject.

In certain embodiments, a hydrate of D3-creatine is administered to thesubject. In particular embodiments, D3-creatine monohydrate isadministered.

The dose of D3-creatine to be administered to the subject is preferablyselected such that the labeled creatine is rapidly absorbed into thebloodstream and spillage of excess label into the urine is minimized.Accordingly, for a human subject the dose of D3-creatine is typically5-250 mgs, such as 20-125 mgs. In particular embodiments, 5, 10, 20, 30,40, 50, 60, 70, 80, 90, or 100 mgs of D3-creatine is administered. Insome embodiments, the dose is adjusted based on the gender of thesubject. Thus, in certain embodiments, the subject is female and 10-50,such as 20-40, or more particularly, 30 mg of D3-creatine isadministered to the subject. In other embodiments, the subject is maleand 40-80 mg, such as 50-70, or more particularly, 60 mg or 70 mg ofD3-creatine is administered to the subject.

Pharmaceutical formulations adapted for oral administration may bepresented as discrete units such as capsules or tablets; powders orgranules; solutions or suspensions, each with aqueous or non-aqueousliquids; edible foams or whips; or oil-in-water liquid emulsions orwater-in-oil liquid emulsions. For instance, for oral administration inthe form of a tablet or capsule, the active drug component may becombined with an oral, non-toxic pharmaceutically acceptable inertcarrier such as ethanol, glycerol, water, and the like. Generally,powders are prepared by comminuting the compound to a suitable fine sizeand mixing with an appropriate pharmaceutical carrier such as an ediblecarbohydrate, as, for example, starch or mannitol. Flavorings,preservatives, dispersing agents, and coloring agents may also bepresent.

Capsules can be made by preparing a powder, liquid, or suspensionmixture and encapsulating with gelatin or some other appropriate shellmaterial. Glidants and lubricants such as colloidal silica, talc,magnesium stearate, calcium stearate, or solid polyethylene glycol maybe added to the mixture before the encapsulation. A disintegrating orsolubilizing agent such as agar-agar, calcium carbonate or sodiumcarbonate may also be added to improve the availability of themedicament when the capsule is ingested. Moreover, when desired ornecessary, suitable binders, lubricants, disintegrating agents, andcoloring agents may also be incorporated into the mixture. Examples ofsuitable binders include starch, gelatin, natural sugars such as glucoseor beta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth, or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes, and the like. Lubricants useful in thesedosage forms include, for example, sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride,and the like. Disintegrators include, without limitation, starch, methylcellulose, agar, bentonite, xanthan gum, and the like.

Tablets can be formulated, for example, by preparing a powder mixture,granulating or slugging, adding a lubricant and disintegrant, andpressing into tablets. A powder mixture may be prepared by mixing thecompound, suitably comminuted, with a diluent or base as describedabove. Optional ingredients include binders such ascarboxyrnethylcellulose, aliginates, gelatins, or polyvinyl pyrrolidone,solution retardants such as paraffin, resorption accelerators such as aquaternary salt, and/or absorption agents such as bentonite, kaolin, ordicalcium phosphate. The powder mixture may be wet-granulated with abinder such as syrup, starch paste, acadia mucilage or solutions ofcellulosic or polymeric materials, and forcing through a screen. As analternative to granulating, the powder mixture may be run through thetablet machine and the result is imperfectly formed slugs broken intogranules. The granules may be lubricated to prevent sticking to thetablet forming dies by means of the addition of stearic acid, a stearatesalt, talc or mineral oil. The lubricated mixture is then compressedinto tablets. The compounds of the present invention may also becombined with a free flowing inert carrier and compressed into tabletsdirectly without going through the granulating or slugging steps. Aclear or opaque protective coating consisting of a sealing coat ofshellac, a coating of sugar or polymeric material, and a polish coatingof wax may be provided. Dyestuffs may be added to these coatings todistinguish different unit dosages.

Oral fluids such as solutions, syrups, and elixirs may be prepared indosage unit form so that a given quantity contains a predeterminedamount of the compound. Syrups may be prepared, for example, bydissolving the compound in a suitably flavored aqueous solution, whileelixirs are prepared through the use of a non-toxic alcoholic vehicle.Suspensions may be formulated generally by dispersing the compound in anon-toxic vehicle. Solubilizers and emulsifiers such as ethoxylatedisostearyl alcohols and polyoxy ethylene sorbitol ethers may be added.Solubilizers that may be used according to the present invention includeCremophor EL, vitamin E, PEG, and Solutol. Preservatives and/or flavoradditives such as peppermint oil, or natural sweeteners, saccharin, orother artificial sweeteners; and the like may also be added.

According to the method, the urine sample in preferably collected afterenrichment levels of D3-creatinine in the urine have reached asteady-state. Thus in one embodiment, at least 6 hours or at least 12hours is allowed to elapse after the administration of the D3-creatinebut prior to the collection of the urine sample. In certain embodiments,at least 24 hours is allowed to elapse. In particular embodiments, atleast 36 hours, at least 48 hours, at least 60 hours, or at least 72hours are allowed to elapse after the administration of the D3-creatineand before the collection of the urine sample.

The invention also encompasses certain improved analytic methods fordetecting creatinine and D3-creatinine in urine samples. Specifically,the invention provides for the detection of creatinine and D3-creatininein urine samples by HPLC/MS, particularly HPLC/MS/MS. However, alternatemethods know in the art may also be used to detect creatinine and/or D3creatinine in urine samples. Such methods include direct or indirectcalorimetric measurements, the Jaffémethod, enzymatic degradationanalysis, or derivatization of the creatinine followed by GC/MS analysisof HPLC with fluorescence detection.

Thus in one aspect, the invention provides a method of determining theconcentration of creatinine in a biological sample from a subject, saidmethod comprising the steps of:

(a) obtaining a biological sample from the subject;

(b) analyzing the biological sample to determine the peak area of thecreatinine M+2 isotope peak for the biological sample;

(c) comparing the peak area determined in step (b) to a calibrationcurve generated using D3-creatinine to determine the concentration ofthe creatinine M+2 isotope in the biological sample;

(d) dividing the concentration obtained in step (c) by a dilutionfactor, where the dilution factor is the ratio of the concentration ofcreatinine M+2 to the concentration of creatinine M+0 in the biologicalsample.

The biological sample may be any appropriate sample including, but notlimited to, urine, blood, serum, plasma, or tissue. In one particularembodiment, the biological sample is a urine sample. In anotherparticular embodiment, the biological sample is a blood sample.

In a preferred embodiment, the peak area of the creatinine M+2 isotopepeak is determined using liquid chromatography/mass spectroscopy(LC/MS/MS).

In one embodiment, the dilution factor is 0.0002142±0.0000214. Moreparticularly, the dilution factor is 0.0002142±0.00001, such as0.0002142±0.000005.

The methods of the invention are useful for diagnosing and monitoringmedical conditions associated with changes in total body skeletal musclemass. Examples of medical conditions in which loss of muscle mass playsan important role in function, performance status, or survival include,but are not limited to frailty and sarcopenia in the elderly; cachexia(e.g., associated with cancer, chronic obstructive pulmonary disease(COPD), heart failure, HIV-infection, tuberculosis, end stage renaldisease (ESRD); muscle wasting associated with HIV therapy, disordersinvolving mobility disability (e.g., arthritis, chronic lung disease);neuromuscular diseases (e.g., stroke, amyotrophic lateral sclerosis);rehabilitation after trauma, surgery (including hip-replacementsurgery), medical illnesses or other conditions requiring bed-rest;recovery from catabolic illnesses such as infectious or neoplasticconditions; metabolic or hormonal disorders (e.g., diabetes mellitus,hypogonadal states, thyroid disease); response to medications (e.g.,glucocorticoids, thyroid hormone); malnutrition or voluntary weightloss. The claimed methods are also useful in sports-related assessmentsof total body skeletal muscle mass.

The methods of the invention are also useful for screening testcompounds to identify therapeutic compounds that increase total bodyskeletal muscle mass. According to this embodiment, the total bodyskeletal mass of a subject is measured according to the method beforeand after a test compound is administered to the subject. The assessmentof total body skeletal muscle mass can be repeated at appropriateintervals to monitor the effect of the test compound on total bodyskeletal muscle mass.

EXPERIMENTAL Use of the D3-Creatine Tracer Dilution Method to DetermineTotal Body Skeletal Muscle Mass in a Pre-Clinical Model

A dose of 0.475 mg D3-creatine per rat was determined to be rapidly andcompletely absorbed and reach the systemic circulation with minimalurinary spillage, such that >99% of the D3-creatine tracer dose shouldbe available to equilibrate with the body creatine pool.

The creatine dilution method was then used to determine urinaryD3-creatine enrichment and the time to isotopic steady state in growingrats. In a cross-sectional study, a single oral dose of 0.475 mgD3-creatine per rat was given to two groups of rats, 9 and 17 weeks ofage, and urine was collected at 24, 48, and 72 hour time points afterdosing. As expected, the larger, older rats had lower urinaryD3-creatinine enrichment (expressed as mole percent excess, MPE) at alltime points than the younger, smaller rats, reflecting greater dilutionof the D3-creatine tracer in the total body creatine pool. For both agegroups, urinary enrichment was highest at 24 h and stable between 48 and72 h, indicating isotopic steady state was achieved between 24 and 48 hafter the tracer D3-creatine dose. (FIG. 1A).

Total body creatine pool size was then calculated using a formula fordetermination of pool size based on enrichment of a tracer, assuming asingle creatine pool (Wolfe and Chinkes (2005) Calculation of substratekinetics: Single-pool model. 2nd ed. Isotope tracers in metabolicresearch. Hoboken, N.J.: John Wiley & Sons, Inc. 21-9): the D3-creatinedose (0.475 mg) was divided by the D3-creatinine enrichment (MPE/100).FIG. 1B shows the total body creatine pool sizes calculated from urinaryenrichment 72 h after the tracer dose for the 9 and 17 week-old ratgroups and indicates the creatine pool size for the larger, older ratsis significantly larger than for the smaller, younger rats.

The day before giving the tracer dose of D3-creatine, lean body mass(LBM) in all rats was assessed by either quantitative magnetic resonance(QMR) or DEXA. FIG. 2 shows that after accounting for age effect, LBM byQMR and creatine pool size are significantly correlated. LBM by QMR andcreatine pool size are also significantly correlated within each agegroup, and LBM by DEXA and creatine pool size are significantlycorrelated within the 17 week-old age group (FIG. 3).

In a second cross-sectional study, an older rat age group (still withinthe rat growth phase of 22 weeks of age) was treated once dailysubcutaneously with either saline vehicle, or dexamethasone to induceskeletal muscle atrophy for 2 weeks prior the administration ofD3-creatine. As with the first cross-sectional study with 9 and 17week-old rats, isotopic steady state was reached between 48 and 72 h.

Compared to vehicle-treated controls, dexamethasone induced asignificant reduction in LBM (353±32 vs. 459±45 g, P<0.001) and asignificant reduction in total body creatine pool size (1216±227 vs.1853±228 mg, P<0.001). As in the first study, LBM and creatine pool sizewere significantly correlated within the two individual treatment groups(FIG. 4).

FIG. 5 show the correlation between LBM and creatine pool size for all40 rats used in the two cross-sectional studies (r=0.95; P<0.001).

Use of the D3-Creatine Tracer Dilution Method to Determine Total BodySkeletal Muscle Mass in Human Subjects

Human subjects are orally administered a single dose of 30, 60, or 100rugs of D3 creatine-monohydrate. Urine samples are then collected 1, 2,3, 4, 5,or 6 days after administration of the D3-creatine monohydratedose.

Urine pharmacokinetic analyses for each collection interval may includequantitation of MPE ratio by IRMS, ratio of deuterium-labeledcreatine+deuterium−labeled creatinine to total creatine+total creatinineby LCMS, total creatinine, creatine pool size, and % ofdeuterium-labeled creatine dose excreted in urine.

Steady-state enrichment (MPE) can be assessed both visually and from theestimate of the slope from the linear regression of enrichment (MPE) vstime (midpoint of each urine collection interval). A mixed effect ANOVAmodel can be fit with time (continuous variable) as a fixed effect andsubject as a random effect. The coefficient for the slope of the timeeffect can be used to evaluate steady-state. The 90% confidenceintervals for the slope can be calculated.

Creatine pool size can be estimated once steady-state enrichment hasbeen achieved a for each collection interval during steady-stateaccording to the formula:

[Amount of D3 Cr dosed(g)−total Amount of urinary D3Cr(0−t)(g)]/enrichment ratio(t) where t is the urine collection intervalduring steady-state.

Muscle mass can be estimated from the creatine pool size by assumingthat the creatine concentration is 4.3 g/kg of whole wet muscle mass(WWM) (Kreisberg (1970) J Appl Physiol 28:264-7).

Muscle mass=creatine pool size/Cr concentration in muscle

Creatine pool size can also be estimated by total urine creatinine(moles/day) divided by K (1/day).

The excretion rate constant (K) can be estimated using a rate excretionmethod by estimating the declining slope of the line for the log of theamount of D3-creatine in urine collection interval vs. time (midpoint ofthat urine collection interval) for each collection interval over time.This estimate of K can be used in calculating creatine pool size from 24hr urinary creatinine excretion rather than using an estimate ofturnover form the literature.

Analytic Methods for Quantitating D3-Creatine and D3-Creatinine in UrineSamples From Clinical Subjects

Reference Standards of D3-Creatine monohydrate and D3-creatinine werepurchased from C/D/N Isotopes, Montreal Canada.

HPLC-MS/MS Analysis

The separation of D3-creatine was carried out using an Acquity UPLC(Waters Corp., Milford, Mass.) equipped with a Zorbax Hilic Plus silicaanalytical column (50×2.1 mm, Rapid Resolution HD 1.8 μ, Agilent Corp.,Santa Clara Calif.). Injection volume is typically 8 μL.

Mobile phase A (MP A) consisted of 10 mM ammonium formate in water andmobile phase B is acetonitrile. Gradient chromatography was employedwith initial mobile phase composition of 2% 10 mM ammonium formate witha flow rate of 0.7 mL/min. This was held for 0.5 minute and then alinear gradient to 50% MPA was achieved at 2.3 minutes. This wasimmediately increased to 80% and held for 0.4 minutes and then returnedto starting conditions at 2.9 minutes. The total run time was 3.5minutes. This gradient allowed baseline separation of the D3-creatinefrom interfering compounds.

The detection of D3-creatine was carried out using a Sciex API5000(Applied Biosystems, Foster City, Calif.). The HPLC system was connectedto the API5000 through a turbo ion spray source operating in positiveionization mode using the following parameters: ionization temperatureof 650° C., ionspray voltage of 2500 V, curtain gas setting of 45 (N₂),nebulizer gas setting was 65 (N₂), drying gas setting was 70 (N₂),collision gas setting of 3 (N₂). All other mass spectrometer parameterswere optimized for the individual transitions. The following iontransitions (MRM) were acquired: D3-creatine is m/z=135 to m/z=47 with atypical retention time of 1.99 min. The creatine standard is monitoredwith an ion transition of m/z=139 to m/z=50 with a typical retentiontime of 1.99 min.

The separation of the creatinine and D3-creatinine analytes were carriedout using an Acquity FPLC (Waters Corp., Milford, Mass.) equipped with aZorbax Hilic Plus silica analytical column , dimensions of 50×2.1 mm(Rapid Resolution HD 1.8 μ, Agilent Corp., Santa Clara Calif.).Injection volume was typically 5 μL.

Mobile phase A consisted of 5 mM ammonium formate and mobile phase B wasacetonitrile. Gradient chromatography was employed with initial mobilephase composition of 2% 5 mM ammonium formate with a flow rate of 0.7mL/min. This was held for 0.4 minute and then a linear gradient to 40%MPA was achieved at 2.1 minutes. This was immediately increased to 50%at 2.2 minutes and held for 0.4 minutes and then returned to startingconditions at 2.7 minutes. The total run time was 3.5 minutes. Thisgradient allowed baseline separation of the d3-creatinine and creatininefrom interfering compounds.

The detection of the creatinine and D3-creatinine analytes was carriedout using a Sciex API5000 (Applied Biosystems, Foster City, Calif.). TheHPLC system was connected to the API5000 through a turbo ion spraysource operating in positive ionization mode using the followingparameters: ionization temperature of 350° C., ionspray voltage of 5500V, curtain gas setting of 45 (N₂), nebulizer gas setting was 60 (N₂),drying gas setting was 65 (N₂), collision gas setting of 3 (N₂). Allother mass spectrometer parameters were optimized for the individualtransitions. The following ion transitions (MRM) were acquired:D3-creatinine is m/z=117 to m/z=47 and for creatinine (M+2 isotope) wasm/z=116 to m/z=44 with a typical retention time of 1.5 min. The creatinestandard is monitored with an ion transition of m/z=121 to m/z=51 with atypical retention time of 1.5 min. For creatinine, the M+2 isotopeversion was acquired to avoid diluting the sample with buffer.

Endogenous creatinine concentration values are determined in human urineclinical samples using a D3-creatinine calibration standard curve. TheD3-creatinine isotope behaves similarly to creatinine throughout theextraction and HPLC-MS/MS procedures, thus allowing clean urine matrixto prepare standards and QC samples.

The amount of endogenous creatinine (m/z=114) in the human clinicalsamples is much greater (˜1000 fold) than the levels of D3-creatinine.Therefore, instead of diluting the sample, the M+2 isotope of creatinine(m/z=116) will be monitored, thus allowing the simultaneous measurementof creatinine and D3-creatinine from one sample analysis. The MRM of(M+2) endogenous creatinine (116/44) is monitored. A correction factorthat represents the ratio of the MRM of 116/44 to 114/44, is used tocorrect the calculated concentrations determined from the d3-creatininecalibration curve. The isotope ratio (M+2) MRM/(M+0) MRM or correctionfactor is 0.00286. Therefore, the amount of D3-creatinine, which wouldcome from the D3 creatine dose and the endogenous creatinine, can bequantitated from the single D3-creatinine calibration curve.

EXAMPLE

Chemical and Reagents: Acetonitrile and Water (all HPLC grade or better)purchased from Sigma Aldrich (St. Louis, Mo.). Ammonium Formatepurchased from Sigma Aldrich (St. Louis, Mo.). Reference Standards ofd3-Creatine (monohydrate) and d3-creatinine were purchased from CDNIsotopes, Montreal Canada.

Stock solutions of d3-creatine and d3-creatinine are prepared at 1.0mg/mL in water and confirmation of equivalence is performed. Dilutesolutions ranging from 0.1 μg/mL to 100 μg/mL and 0.2 μg/mL to 200 μg/mLare prepared in water and used to prepare calibration standards andquality control (QC) samples in human urine for d3-creatine andd3-creatinine, respectively. Isotopically labelled internal standardsfor creatine (SIL) ¹³C₃ ²H₃ ¹⁵N₁creatine) and creatinine (SIL) (¹³C₃ ²H₄¹⁵ N₁-creatinine) are prepared at 1.0 mg/mL in water. Dilute solutionsof these are prepared at 500 ng/mL in acetonitrile and used as anextraction solvent for the urine standards, quality controls and studysamples.

Sample Preparation: (d3-creatine, creatinine and d3-creatinine in urine)A 200 μL aliquot of the internal standard working solution (500 ng/mL)in acetonitrile is added to each well, except double blank samples,acetonitrile is added. A 40 μL aliquot of sample, standard or QC istransferred to the appropriate wells in the plate containing the SIL.The plate is sealed and vortex mixed for approximately 3 minutes. Theplate is centrifuged at approximately 3000 g for 5 minutes. Supernatantis transferred to a clean 96 well plate and then injected onto theHPLC-MS/MS system for analysis. D3-creatine and d3-creatinine areanalyzed from separate human urine samples.

HPLC-MS/MS Analysis

The separation of d3-creatine, d3-creatinine and creatinine is carriedout using an Acquity UPLC (Waters Corp., Milford, Mass.) equipped with aAgilent Zorbax Hilic Plus silica analytical column , dimensions of50×2.1 mm (Rapid Resolution HD 1.8μ, Agilent Corp., Santa Clara Calif.).Injection volume is typically 2 μL.

D3-creatine: mobile phase A consists of 10 mM ammonium formate andmobile phase B is acetonitrile. Gradient chromatography is employed withinitial mobile phase composition at 2% 10 mM ammonium formate with aflow rate of 0.7 mL/min. This is held for 0.5 minute and then a lineargradient to 50% MPA is achieved at 2.3 minutes. This is increased to 80%over 0.2 minutes and held for 0.4 minutes and then returned to startingconditions at 3.0 minutes. The total run time is 3.5 minutes.

The detection of d3-creatine is carried out using a Sciex API5000(Applied Biosystems, Foster City, Calif.). The HPLC system is connectedto the API5000 through a turbo ion spray source operating in positiveionization mode using the following parameters: ionization temperatureof 650° C., ionspray voltage of 2500 V, curtain gas setting of 45 (N₂),nebulizer gas setting is 65 (N₂), drying gas setting is 70 (N₂),collision gas setting of 3 (N₂). All other mass spectrometer parametersare optimized for the individual transitions. The following iontransitions (MRM) are acquired: d3-creatine is m/z=135 to m/z=47 with atypical retention time of 2 min. The SIL is monitored with an iontransition of m/z=139 to m/z=50 with a typical retention time of 2 min.

D3-creatinine: mobile phase A consisted of 5 mM ammonium formate, andmobile phase B is acetonitrile. Gradient chromatography is employed withinitial mobile phase composition at 2% 5 mM ammonium formate with a flowrate of 0.7 mL/min. This is held for 0.4 minute and then a lineargradient to 60% acetonitrile is achieved at 2.1 minutes. This isimmediately increased to 50% acetonitrile and held for 0.4 minutes andthen returned to starting conditions at 2.7 minutes. The total run timeis 3.5 minutes.

The detection of the creatinine and d3-creatinine analytes is carriedout using a Sciex API5000 (Applied Biosystems, Foster City, Calif.). TheHPLC system was connected to the API5000 through a turbo ion spraysource operating in positive ionization mode using the followingparameters: ionization temperature of 350° C., ionspray voltage of 5500V, curtain gas setting of 45 (N₂), nebulizer gas setting was 60 (N₂),drying gas setting was 65 (N₂), collision gas setting of 3 (N₂). Allother mass spectrometer parameters are optimized for the individualtransitions. The following ion transitions (MRM) are acquired:d3-creatinine is m/z=117 to m/z=47 and for creatinine (M+2 isotope) ism/z=116 to m/z=44 with a typical retention time of 1.5 min. The SIL ismonitored with an ion transition of m/z=121 to m/z=51 with a typicalretention time of 1.5 min. For creatinine, the M+2 isotope MRM isacquired to avoid diluting the sample with a surrogate matrix (acreatinine free control urine is not available). These isotopes willbehave similarly throughout the extraction and HPLC-MS/MS procedures,thus allowing clean urine matrix to prepare standards and QC samples aswell as allowing for the quantification of endogenous creatinine using acalibration curve that was generated from the deuterated form ofcreatinine. Therefore, the amount of d3-creatinine and the endogenouscreatinine, can be quantitated from the single d3-creatinine calibrationcurve.

HPLC-MS/MS data were acquired and processed (integrated) using Analyst™software (Version 1.4.2, MDS Sciex, Canada). A calibration plot of arearatio versus d3-creatinine concentration was constructed and a weighted1/x² linear regression applied to the data.

RESULTS

To perform bioanalytical quantification of biomarkers using LC/MS/MS, asurrogate matrix or a surrogate analyte must be used. In this assay,human urine can be used since d3-creatinine is not found endogenouslyand the quantification of creatinine can be determined from thed3-creatinine calibration curve. The equivalency of d3-creatinine andcreatinine is shown.

D3-Creatinine and Creatinine Equivalence Determination

A number of experiments were performed in order to verify that d3creatinine can be used as a surrogate analyte to quantitate creatinineand that the MRM transition of 116/44 (M+2) can be used with the isotoperatio correction factor.

To confirm that d3 creatinine can be used as a surrogate analyte forcreatinine; two concentration levels of creatinine and d3 creatinineneat standard solutions were prepared to show equivalent LC-MS/MSresponse. The peak areas of 200 ng/mL and 40 ng/mL of both creatinineand d3 creatinine standard solutions were compared using the MRMtransitions of 114/44 and 117/47, respectively. The results showed thatthe two solutions gave equivalent responses with mean percent differenceand percent CV of less than 7.5%. See Table 1.

TABLE 1 D3 creatinine and creatinine equivalence using LC/MS/MS CRN vsPercent Std Creatinine d3 CRN of D3 (ng/mL) (MRM of 114/44) % differenceResponse d3-Creatinine (MRM of 117/44) 40 791648 717010 10.4 90.6 40804513 780182 3.1 97.0 40 774228 717528 7.9 92.7 40 776144 823064 −5.7106.0 40 766927 828642 −7.4 108.0 40 741290 758937 −2.3 102.4 Mean 1.099.4 % CV 7.2 d3-Creatinine (MRM of 117/47) 200 3296195 3336107 −1.2101.2 200 3469440 3325274 4.3 95.8 200 3416181 3428709 −0.4 100.4 2003363696 3185389 5.6 94.7 200 3335259 3390463 −1.6 101.7 200 32557993321365 −2.0 102.0 Mean 0.8 99.3 % CV 3.2

These results show that d3 creatinine and creatinine give equivalentLC/MS/MS responses and d3-creatinine can be used as a surrogate analytefor creatinine. This is not surprising since deuterated compounds areused routinely as stable label internal standards, in regulatedenvironments to validate assays. These deuterated standards have beenshown to correct LC/MS/MS response of analyte from matrix effects aswell as other extraction and chromatographic related effects. Since theonly difference is an extra proton at three hydrogen atoms on the methylgroup, we would expect the two compounds to behave almost identicallythroughout the extraction, chromatographic separation and mass spectraldetection.

Determination of Isotope Ratio

This method is used to determine the amount of d3 creatinine in humanurine that has been converted from a dose of d3 creatine. Additionally,the amount of endogenous creatinine will be determined using the d3creatinine standard curve. The amount of endogenous creatinine is muchgreater (˜1000 fold) than the levels of d3-creatinine in the humanclinical urine samples, therefore instead of diluting the sample, theM+2 isotope of creatinine will be monitored. This will allow thesimultaneous measurement of creatinine and d3-creatinine from one sampleusing a urine matrix calibration curve. The peak area of the MRM of(M+2) endogenous creatinine (116/44) is monitored along with the d3creatinine MRM of 117/47. A correction factor that represents the ratioof the MRM of 116/44 to 114/44, is used to correct the calculatedconcentrations determined from the d3-creatinine calibration curve.

The isotope ratio (response ratio) or difference in peak area responsefrom the naturally abundant form of creatinine (M+0) or m/z=114 to themuch less abundant form of creatinine (M+2) or m/z=116 is calculatedexperimentally. The isotope ratio is determined using two differentexperimental procedures. The original experimental design uses onestandard concentration, a 200 ng/mL creatinine solution (Table 2a).

The peak area of the creatinine is monitored at both the M+0 and M+2 MRMtransitions (114/44 and 116/44), respectively. One solution was used toreduce variation which may occur from separate injections andpreparation of separate solutions. This concentration is chosen becauseit allows the peak area of both MRMs to be in the detector range, andwith adequate signal to noise for the smaller peak. However, somevariability in the day to day measurements is observed (±10%) as shownin Table 3.

Therefore, an additional experiment to generate this response ratio wasperformed. In the second approach, the response ratio is experimentallydetermined using two separate solutions. A separate solution for eachMRM transition is prepared which gives peak areas that are closer inmagnitude to each other. A 10 ng/mL solution of creatinine is used toacquire the MRM transition of 114/144 and a 500 ng/mL solution is usedto acquire the MRM transition of 116/44. These solutions are injected onthe LC/MS/MS system in replicates of 10 and the mean peak area ratio(PAR) for each solution is determined. The response ratio is thencalculated by dividing the mean PAR of 116/44 by the corrected PAR of114/44. In order to compare the PARs from the two MRMs, the PAR from the10 ng/mL solutions is multiplied by 50 (since 500 ng/mL is 50 timeslarger than the 10 ng/mL), an example is shown in Table 2b. This allowsthe peak area of both solutions to be closer in value and potentiallyeliminating errors associated with integrating peaks with vastlydifferent signal to noise values.

TABLE 2 Creatinine Response Ratio (M + 2/M + 0) Determination usingLC/MS/MS 2a. Determined using a single creatinine standard solution PeakArea Ratio STD Creatinine (M + 2) Creatinine (M + 0) Response (ng/mL)MRM of 116/44 MRM of 114/44 Ratio 200 0.0209 9.19 0.00227 200 0.02169.42 0.00229 200 0.0195 9.53 0.00205 200 0.0208 9.42 0.00221 200 0.02029.37 0.00216 200 0.0199 9.46 0.00210 200 0.0188 9.22 0.00204 200 0.02019.64 0.00209 200 0.0202 9.33 0.00217 200 0.0188 9.49 0.00198 200 0.02009.45 0.00212 200 0.0198 9.32 0.00212 Mean 0.0201 9.4 0.00213 % CV 4.051.35 3.10 2b. Determined using separate creatinine concentrations PeakArea Ratio Creatinine (M + 2) Creatinine Creatinine MRM of (M + 0) (M +0)* Response 116/44 MRM of 114/44 MRM of 114/44 Ratio 0.0460 0.4240 21.20.00217 0.0467 0.4240 21.2 0.00220 0.0466 0.3990 20.0 0.00234 0.04710.4110 20.6 0.00229 0.0477 0.4000 20.0 0.00239 0.0459 0.3850 19.30.00238 0.0453 0.3990 20.0 0.00227 0.0452 0.4000 20.0 0.00226 0.04430.3920 19.6 0.00226 0.0442 0.3780 18.9 0.00234 Mean 0.0459 0.4 20.10.00229 % CV 2.53 3.75 3.75 3.15 * = corrected for concentrationdifference

The corrected peak area ratio would be equivalent to a 500 ng/mLcreatinine standard monitoring the peak area of the MRM transition of114/44.

The isotope ratio (response ratio) was determined on multiple occasionsover a four month time span and on two different triple quadrapoleinstruments. The mean of these nine values was determined and theinverse of this response ratio is the dilution factor used to correctthe creatinine values in the LIMS system. See Table 3.

TABLE 3 Summary of Response Ratio (M + 2/M + 0) Determined usingLC/MS/MS Date Response Ratio Instrument Name 22-Nov-11 0.00206 RTP1229-Nov-11 0.00213 RTP12 AM 7-Dec-11 0.00193 RTP12 PM 7-Dec-11 0.00193RTP12 * 14-Jan-12 0.00221 RTP12 * 16-Jan-12 0.00229 RTP12 * 17-Jan-120.00195 RTP12 * 7-Feb-12 0.00229 RTP12 * 10-Feb-12 0.00249 RTP52 Mean0.002142 % CV 9.09 *= performed using two concentrations of creatinine

This experimentally determined response ratio is used to correct peakareas of creatinine M+2 (MRM of 116/44) and these corrected peak areasof creatinine were compared to peak areas run for the same concentrationof d3 creatinine standard (MRM of 117/47). The comparison of thecorrected creatinine peak area to the peak area obtained from the d3creatinine standards gave equivalent responses with percent differenceand percent CV of less than 10%. See Table 4.

TABLE 4 Creatinine (M + 2) Response Corrected using Response Ratio CRNCRN M + 2* d3 CRN STD (MRM 116/44) Corrected as to (MRM 117/47) Percent(ng/mL) M + 2 M + 0 Peak Area Difference 200 7856.2 3667693.7 3599411.598.1 200 7422.7 3465312.8 3682013.9 106.3 200 6101.3 2848412.7 3516609.5123.5 200 7490.2 3496825.4 3330922.4 95.3 200 7288.0 3402427.6 3359823.298.7 200 6625.6 3093183.9 3264518.6 105.5 Mean 7130.7 3328976.03458883.2 104.6 % CV 9.0 9.0 4.8 9.8 *= corrected peak area (divided bythe mean response ratio of 0.002142)

1. A method of determining total body skeletal muscle mass in a subject,comprising: (a) orally administering D₃-creatine to a subject, whereinthe D₃-creatine dilutes in the total body skeletal muscle pool ofcreatine and reaches isotopic steady-state in the total body skeletalmuscle pool of creatine; (b) obtaining a biological sample comprisingcreatinine and D₃-creatinine from the subject, wherein the biologicalsample is selected from the group consisting of a blood sample, a serumsample, a plasma sample, and a tissue sample; (c) detecting creatinineand D₃-creatinine in the biological sample by a method selected from thegroup consisting of HPLC/MS, HPLC/MS/MS, LCMS, LC/MS/MS, and isotoperatio mass spectrometry (IRMS); (d) measuring enrichment ratio ofD₃-creatinine in the biological sample at a time t based on thecreatinine and D₃-creatinine detected in (c); (e) measuring a totalamount of urinary D₃-creatinine from administration of the D₃-creatineto the time t; (f) determining total-body creatine pool size in thesubject by the formula ${\frac{\begin{matrix}\left\lbrack {{{Amount}\mspace{14mu} {of}\mspace{14mu} D_{3}\text{-}{creatine}\mspace{14mu} {dosed}\mspace{14mu} (g)} -} \right. \\\left. {{Total}\mspace{14mu} {amount}\mspace{14mu} {of}\mspace{14mu} {urinary}\mspace{14mu} D_{3}\text{-}{creatine}\mspace{14mu} \left( {o - t} \right)(g)} \right\rbrack\end{matrix}}{{enrichment}\mspace{14mu} {ratio}\mspace{14mu} (t)} = {{total}\text{-}{body}\mspace{14mu} {creatine}\mspace{14mu} {pool}\mspace{14mu} {size}}};$and (g) determining total body skeletal muscle mass in the subject basedon the formula${{Total}\mspace{14mu} {body}\mspace{14mu} {skeletal}\mspace{14mu} {muscle}\mspace{14mu} {mass}} = {\left( \frac{{the}\mspace{14mu} {total}\text{-}{body}\mspace{14mu} {creatine}\mspace{14mu} {pool}\mspace{14mu} {size}}{{creatine}\mspace{14mu} {concentration}\mspace{14mu} {in}\mspace{14mu} {skeletal}\mspace{14mu} {muscle}} \right).}$2. (canceled)
 3. The method of claim 1, wherein the biological sample isa blood sample.
 4. The method of claim 1, wherein 5-250 mg D₃-creatineor a salt or hydrate thereof are administered.
 5. The method of claim 1,wherein the D₃-creatine administered is a hydrate of D₃-creatine.
 6. Themethod of claim 5, wherein the D₃-creatine is D₃-creatine monohydrate.7. The method of claim 1, wherein the biological sample is obtained atleast 24 hours after administration of the D₃-creatine.
 8. The method ofclaim 7, wherein the biological sample is obtained at least 36 hoursafter administration of the D₃-creatine.
 9. The method of claim 7,wherein the biological sample is obtained at least 48 hours afteradministration of the D₃-creatine.
 10. The method of claim 7, whereinthe biological sample is obtained at least 60 hours after administrationof the D₃-creatine.
 11. The method of claim 7, wherein the biologicalsample is obtained at least 72 hours after administration of theD₃-creatine.
 12. The method of claim 1, wherein the D₃-creatine isadministered to the subject such that the spillage of D₃-creatine intothe urine is minimized, wherein greater than 99% of the administeredD₃-creatine dilutes in the total body skeletal muscle pool of creatineand reaches isotopic steady-state in the total body skeletal muscle poolof creatine.
 13. The method of claim 1, wherein the creatineconcentration in skeletal muscle is 4.3 g/kg.
 14. The method of claim 1,wherein the biological sample is selected from the group consisting of ablood sample, a serum sample, and a plasma sample